DWARF4 accumulation in root tips is enhanced via blue light perception by cryptochromes

Brassinosteroid (BR) signalling is known to be coordinated with light signalling in above ground tissue. Many studies focusing on the shade avoidance response in above ground tissue or hypocotyl elongation in darkness have revealed the contribution of the BR signalling pathway to these processes. We previously analysed the expression of DWARF 4 (DWF4), a key BR biosynthesis enzyme, and revealed that light perception in above ground tissues triggered DWF4 accumulation in root tips. To determine the required wavelength of light and photoreceptors responsible for this regulation, we studied DWF4‐GUS marker plants grown in several monochromatic light conditions. We revealed that monochromatic blue LED light could induce DWF4 accumulation in primary root tips and root growth as much as white light, whereas monochromatic red LED could not. Consistent with this, a cryptochrome1/2 double mutant showed retarded root growth under white light whereas a phytochromeA/B double mutant did not. Taken together, our data strongly indicated that blue light signalling was important for DWF4 accumulation in root tips and root growth. Furthermore, DWF4 accumulation patterns in primary root tips were not altered by auxin or sugar treatment. Therefore, we hypothesize that blue light signalling from the shoot tissue is different from auxin and sugar signalling.     

Actinic light and pH effect on the proton pumping of bacteriorhodopsin

The actinic light effect on the bacteriorhodopsin (BR) photocycle kinetics led to the assumption of a cooperative interaction between the photocycling BR molecules. In this paper we report the results of the actinic light effect and pH on the proton release and uptake kinetics. An electrical method is applied to detect proton release and uptake during the photocycle [E. Papp, G. Fricsovszky, J. Photochem. Photobiol. B: Biol. 5 (1990) 321]. The BR photocycle kinetics was also studied by absorption kinetics measurements at 410 nm and the data were analyzed by the local analysis of the M state kinetics [E. Papp, V.H. Ha, Biophys. Chem. 57 (1996) 155]. While at high pH and ionic strength, we found a similar behavior as reported earlier, at low ionic strength the light effect proved to be more complex. The main conclusions are the following: Though the number of BR excited to the photocycle (fraction cycling, fc) goes to saturation with increasing laser pulse energy, the absorbed energy by BR increases linearly with pulse energy. From the local analysis we conclude that the light effect changes the kinetics much earlier, already at the L intermediate state decay. The transient electric signal, caused by proton release and uptake, can be decomposed into two components similarly to the absorption kinetic data of the M intermediate state. The actinic light energy affects mainly the ratio of the two components and the proton movements inside BR while pH has an effect on the kinetics of the proton release and uptake groups at the membrane surface.     

Photochemical conversion of the O-intermediate to 9-cis-retinal-containing products in bacteriorhodopsin films.

The photochemical activity of the O-state was investigated in bacteriorhodopsin (BR) films containing wildtype BR at pH 6.5 in the presence of glycerol. The formation of a photoproduct of O with an absorption maximum at 490 nm and 9-cis-retinal configuration was found. This 490-nm product was named P and shows a slow thermal reaction into a compound with a maximal absorption at 380 nm which was named Q and contains free 9-cis-retinal in the proteins binding site. The photoproducts of O, i.e., P and Q, are very similar, or even identical, to those previously observed in blue membranes. Common to the O-state and blue membrane forms of bacteriorhodopsin is a protonated aspartic acid 85, and we suggest that it is the reduced negative charge around the Schiff base which is responsible for the 9-cis photoisomerization. The release of a proton from aspartic acid 85 is linked to the conversion of the O-state back to the initial state of BR. Therefore the conditions of low proton mobility in BR films containing glycerol favor the accumulation of the O-state. For optical and holographic applications such BR films are very attractive. It is possible to create photoproducts with red light which are thermally stable at room temperature and that can be photochemically erased. Dependent on the light composition both properties can be realized in the same sample material. This feature may bridge the gap between information processing and short-term and long-term storage of information with BR.     

Cryptochrome blue light photoreceptors are activated through interconversion of flavin redox states

Cryptochromes are blue light-sensing photoreceptors found in plants, animals, and humans. They are known to play key roles in the regulation of the circadian clock and in development. However, despite striking structural similarities to photolyase DNA repair enzymes, cryptochromes do not repair double-stranded DNA, and their mechanism of action is unknown. Recently, a blue light-dependent intramolecular electron transfer to the excited state flavin was characterized and proposed as the primary mechanism of light activation. The resulting formation of a stable neutral flavin semiquinone intermediate enables the photoreceptor to absorb green/yellow light (500-630 nm) in addition to blue light in vitro. Here, we demonstrate that Arabidopsis cryptochrome activation by blue light can be inhibited by green light in vivo consistent with a change of the cofactor redox state. We further characterize light-dependent changes in the cryptochrome1 (cry1) protein in living cells, which match photoreduction of the purified cry1 in vitro. These experiments were performed using fluorescence absorption/emission and EPR on whole cells and thereby represent one of the few examples of the active state of a known photoreceptor being monitored in vivo. These results indicate that cry1 activation via blue light initiates formation of a flavosemiquinone signaling state that can be converted by green light to an inactive form. In summary, cryptochrome activation via flavin photoreduction is a reversible mechanism novel to blue light photoreceptors. This photocycle may have adaptive significance for sensing the quality of the light environment in multiple organisms.     

Blue-light-mediated shade avoidance requires combined auxin and brassinosteroid action in Arabidopsis seedlings

Plant growth in dense vegetation can be strongly affected by competition for light between neighbours. These neighbours can not only be detected through phytochrome-mediated perception of a reduced red:far-red ratio, but also through altered blue light fluence rates. A reduction in blue light (low blue) induces a set of phenotypic traits, such as shoot elongation, to consolidate light capture; these are called shade avoidance responses. Here we show that both auxin and brassinosteroids (BR) play an important role in the regulation of enhanced hypocotyl elongation of Arabidopsis seedlings in response to blue light depletion. Only when both hormones are experimentally blocked simultaneously, using mutants and chemical inhibitors, will the response be fully inhibited. Upon exposure to low blue several members of the cell wall modifying XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE (XTH) protein family are regulated as well. Interestingly, auxin and BR each regulate a subset of these XTHs, by which they could regulate cell elongation. We hypothesize that auxin and BR regulate specific XTH genes in a non-redundant and non-synergistic manner during low-blue-induced shade avoidance responses of Arabidopsis seedlings, which explains why both hormones are required for an intact low-blue response.     

A comparison of the second harmonic generation from light-adapted, dark-adapted, blue, and acid purple membrane.

The second order nonlinear polarizability and dipole moment changes upon light excitation of light-adapted bacteriorhodopsin (BR), dark-adapted BR, blue membrane, and acid purple membrane have been measured by second harmonic generation. Our results indicate that the dipole moment changes of the retinal chromophore, delta mu, are very sensitive to both the chromophore structure and protein/chromophore interactions. Delta mu of light-adapted BR is larger than that of dark-adapted BR. The acid-induced formation of the blue membrane results in an increase in the delta mu value, and formation of acid purple membrane, resulting from further reduction of pH to 0, returns the delta mu to that of light-adapted BR. The implications of these findings are discussed.     

Genome-wide analysis of light sensing in Prochlorococcus

Prochlorococcus MED4 has, with a total of only 1,716 annotated protein-coding genes, the most compact genome of a free-living photoautotroph. Although light quality and quantity play an important role in regulating the growth rate of this organism in its natural habitat, the majority of known light-sensing proteins are absent from its genome. To explore the potential for light sensing in this phototroph, we measured its global gene expression pattern in response to different light qualities and quantities by using high-density Affymetrix microarrays. Though seven different conditions were tested, only blue light elicited a strong response. In addition, hierarchical clustering revealed that the responses to high white light and blue light were very similar and different from that of the lower-intensity white light, suggesting that the actual sensing of high light is mediated via a blue-light receptor. Bacterial cryptochromes seem to be good candidates for the blue-light sensors. The existence of a signaling pathway for the redox state of the photosynthetic electron transport chain was suggested by the presence of genes that responded similarly to red and blue light as well as genes that responded to the addition of DCMU [3-(3,4-dichlorophenyl)-1,1-N-N'-dimethylurea], a specific inhibitor of photosystem II-mediated electron transport.     

B-box protein BBX32 integrates light and brassinosteroid signals to inhibit cotyledon opening

Cotyledon opening is a key morphological change that occurs in seedlings during de-etiolation. Brassinosteroids (BRs) inhibit the opening of cotyledons in darkness while light promotes cotyledon opening. The molecular regulation of the interplay between light and BR to regulate cotyledon opening is not well understood. Here, we show the B-box protein BBX32 negatively regulates light signaling and promotes BR signaling to inhibit cotyledon opening in Arabidopsis (Arabidopsis thaliana). BBX32 is highly expressed in the cotyledons of seedlings during de-etiolation. bbx32 and 35S:BBX32 seedlings exhibit enhanced and reduced cotyledon opening, respectively, in response to both light and brassinazole treatment in dark, suggesting that BBX32 mediates cotyledon opening through both light and BR signaling pathways. BBX32 expression is induced by exogenous BR and is upregulated in bzr1-1D (BRASSINAZOLE RESISTANT1-1D). Our in vitro and in vivo interaction studies suggest that BBX32 physically interacts with BZR1. Further, we found that PHYTOCHROME-INTERACTING FACTOR 3 (PIF3) interacts with BBX32 and promotes BR-mediated cotyledon closure. BBX32, BZR1, and PIF3 regulate the expression of common target genes that modulate the opening and closing of cotyledons. Our work suggests BBX32 integrates light and BR signals to regulate cotyledon opening during de-etiolation.     

Actinic light density dependence of the bacteriorhodopsin protocycle.

The photocycle of bacteriorhodopsin (BR) was studied in the 0.3 microsecond to 10 s time interval after excitation, using a wide range of actinic light intensities (10 ns half-duration, 0.06-60 mJ/cm2), at neutral and alkaline pH values. The relative weights of the rapidly and the slowly decaying components of the M intermediate (Mf and M(s), respectively) and the yield of the third millisecond component, N(R,P), are the function of the exciting light intensity (density), while their lifetimes are not. The relative weight of M(s) is found to be a linear function of the portion of the BR molecules undergoing the photocycle. This suggests the existence of a cooperative interaction of the BR molecules arranged in the crystalline purple membrane sheets. Another source of M(s) is also found, which results a nonvanishing relative weight of M(s) even at very weak actinic light density values. The explanation for this may be a branching, or the heterogeneity of BR itself or with its environment. It is shown that the relative weights of the rising and decaying components of the M form(s) do not correlate directly with each other.     

A Blue-shifted Light-driven Proton Pump for Neural Silencing

Ion-transporting rhodopsins are widely utilized as optogenetic tools both for light-induced neural activation and silencing. The most studied representative is Bacteriorhodopsin (BR), which absorbs green/red light (∼570 nm) and functions as a proton pump. Upon photoexcitation, BR induces a hyperpolarization across the membrane, which, if incorporated into a nerve cell, results in its neural silencing. In this study, we show that several residues around the retinal chromophore, which are completely conserved among BR homologs from the archaea, are involved in the spectral tuning in a BR homolog (HwBR) and that the combination mutation causes a large spectral blue shift (λ_max = 498 nm) while preserving the robust pumping activity. Quantum mechanics/molecular mechanics calculations revealed that, compared with the wild type, the β-ionone ring of the chromophore in the mutant is rotated ∼130° because of the lack of steric hindrance between the methyl groups of the retinal and the mutated residues, resulting in the breakage of the π conjugation system on the polyene chain of the retinal. By the same mutations, similar spectral blue shifts are also observed in another BR homolog, archearhodopsin-3 (also called Arch). The color variant of archearhodopsin-3 could be successfully expressed in the neural cells of Caenorhabditis elegans, and illumination with blue light (500 nm) led to the effective locomotory paralysis of the worms. Thus, we successfully produced a blue-shifted proton pump for neural silencing.     

Interactions of green or red light with blue light on the dark closure of Albizzia pinnules

The interactions of green or red light with blue light on the dark closing of Albizzia julibrissin Durazz. pinnules have been investigated. Irradiations at 430, 450 and 470 nm progressively delay dark closing with increasing photon fluence rates. Red or green light alone has no effect. However, when the blue fluence rate is low, both red and green light interact with it and increase the delaying effect of the blue light. When the blue fluence rate is high, green light interacts with it to negate some of the effectiveness of the blue light, while red light has no effect. This is similar to results obtained previously with far-red light. It is suggested that the same unidentified photoreceptor is operating in both the far-red and blue regions. The results also indicate the presence of a blue-only absorbing photoreceptor whose action is increased by phytochrome.     

The photoactivated PYP domain of Rhodospirillum centenum Ppr accelerates the recovery of the bacteriophytochrome domain after white light illumination

Ppr from the purple phototrophic bacterium, Rhodospirillum centenum (also known as Rhodocista centenaria), is a hybrid of photoactive yellow protein (PYP), bacteriophytochrome (Bph), and histidine kinase (HK) domains. The holo-Ppr (containing both chromophores) exhibits characteristic absorption maxima at 435 nm due to the PYP domain and at 400, 642, and 701 nm due to the Bph domain. Illumination of the Ppr with white light causes a bleach of both PYP and Bph absorbance; weak blue light primarily bleaches the PYP, and red light activates only the Bph. When excited by blue light, the PYP domain in Ppr recovers with biphasic kinetics at 445 nm (32% with a lifetime of 3.8 min and the remainder with a lifetime of 46 min); white light primarily results in fast recovery, whereas the 130-residue PYP construct shows only the faster kinetics in both blue and white light. Furthermore, there is a slight red shift of the ground state Bph when the PYP is activated; thus, both spectroscopy and kinetics suggest interdomain communication. When Ppr is illuminated with red light, the recovery of the Bph domain to the dark state is significantly slower than that of PYP and is biphasic (57% of the 701 nm decay has a lifetime of 17 min and the remainder a lifetime of 50 min). However, when illuminated with white light or red followed by blue light, the Bph domain in Ppr recovers to the dark-adapted state in a triphasic fashion, where the fastest phase is similar to that of the fast phase of the PYP domain (in white light, 25% of the 701 nm recovery has a lifetime of approximately 1 min) and the slower phases are like the recovery after red light alone. Apo-holo-Ppr (with the biliverdin chromophore only) recovers with biphasic kinetics similar to those of the slower phases of holo-Ppr when activated by either red or white light. We conclude that the photoactivated PYP domain in Ppr accelerates recovery of the activated Bph domain. Phytochromes can be reversibly switched between Pr and Pfr forms by red and far-red light, but the consequence of a bleaching phytochrome is that it cannot be photoreversed by far-red light. We thus postulate that the function of the PYP domain in Ppr is to act as a blue light switch to reverse the effects of red light on the Bph.     

Effect of riboflavie and cyanide on blue light induction of betacyanin formation in Amamnthus caudatus seedlings

The role of phyto chrome and flavins in blue light induction of betacyanin formation was studied in etiolated, three-day-old Amaranthus caudatus L. seedlings, using the criterion of far-red reversibility and exogenously applied riboflavin and KCN. The effect of riboflavin was studied using high fluence rate blue light (42.7 :nmol m⁻²s⁻¹nm⁻¹ at 450 nm). When present in the incubation medium during illumination, riboflavin promoted the far-red reversibility with short light treatments and suppressed the inductive action of continuous illumiaation. If added after light treatments, it promoted betacyanin formation. The filtration of blue light through the riboflavin solution caused profound changes in light quality without affecting the far-red reversibility after 30 mm illumination. The effect of 1 mM KCN was tested with 70'% lower fluence of blue light. Cyanide caused the suppression of the inductive effect with 5 min blue light, which was accompanied by an enhancement of betacyanin induction by the terminal far-red light pulse. With 30 min blue light, however, it caused the appearance of far-red reversibility. The inductive effect of continuous blue illumination was slightly promoted by this Inhibitor. These results demonstrate that the effect of blue light on the pbyto chrome system is complex, whereas the physiological (inductive) action of the flavin triplet state is limited to low fluence, short blue light treatments.     

菌紫质D96N样品的光暗适应型及其转化机制

通过对菌紫质D96N基因突变形成的薄膜样品在光照下动态光谱的测定和分析,研究了此样品的光适应型、暗适应型特性及其之间的转化机制。实验证实,样品受光照激发后很快从暗适应型（D态）光适应型（B态）进入光循环,经过一系列的光化学中间体到边较稳定的M态。M态经过约1小时热驰豫完全回到光适应型B态,B态再经过24小时缓慢热驰豫完全回到暗适应型D态。B态受光则进入光循环,不受光则转化为D态,从而认为B态是菌紫质光循环路径中一个重要的分支节点。据此,提出了该样品在光照下B,D和M态之间转化的模型。     

Interaction between the spectral photon flux density distributions of light during growth and for measurements in net photosynthetic rates of cucumber leaves

The net photosynthetic rate of a leaf becomes acclimated to the plant's environment during growth. These rates are often measured, evaluated and compared among leaves of plants grown under different light conditions. In this study, we compared net photosynthetic rates of cucumber leaves grown under white light‐emitting diode (LED) light without and with supplemental far‐red (FR) LED light (W‐ and WFR‐leaves, respectively) under three different measuring light (ML) conditions: their respective growth light (GL), artificial sunlight (AS) and blue and red (BR) light. The difference in the measured photosynthetic rates between W‐ and WFR‐leaves was greater under BR than under GL and AS. In other words, an interaction between supplemental FR light during growth and the spectral photon flux density distribution (SPD) of ML affected the measured net photosynthetic rates. We showed that the comparison and evaluation of leaf photosynthetic rates and characteristics can be biased depending on the SPD of ML, especially for plants grown under different photon flux densities in the FR waveband. We also investigated the mechanism of the interaction. We confirmed that the distribution of excitation energy between the two photosystems (PSs) changed in response to the SPD of GL, and that this change resulted in the interaction, as suggested in previous reports. However, changes in PS stoichiometry could not completely explain the adjustment in excitation energy distribution observed in this study, suggesting that other mechanisms may be involved in the interaction.     

Blue light signalling in chloroplast movements

Chloroplast movements are among the mechanisms allowing plants to cope with changes in their environment. Chloroplasts accumulate at illuminated cell areas under weak light while they avoid areas exposed to strong light. These directional responses may be controlled by blue and/or red light, depending on the plant group. In terrestrial angiosperms only the blue light perceived by phototropins is active. The last decade has seen a rapid development of studies on the mechanism of directional chloroplast movements, which started with an identification of the photoreceptors. A forward genetic approach has been used to identify the components which control chloroplast movements. This review summarizes the current state of research into the signalling pathways which lead to chloroplast responses. First, the molecular properties of phototropins are presented, followed by a characterization both of proteins which are active downstream of phototropins and of secondary messengers. Finally, cross-talk between light signalling involved in chloroplast movements and other signalling pathways is discussed.     

Reception of far-ultraviolet light in Phycomyces: antagonistic interaction with blue and red light

Phototropism experiments were done with sporangiophores of the fungus Phycomyces blakesleeanus to characterize the interaction between far-UV, blue and red light. Far-UV light elicits negative phototropism (bending away from the light source) while blue light elicits positive phototropism (bending toward the light source). In contrast, red light above 600 nm is phototropically inert. Phototropism was analyzed with light regimens of bilateral or unilateral irradiation with far-UV and blue light. Under bilateral irradiation, in which the two light sources were facing each other, blue light partially inhibited the far-UV-elicited phototropism. A fluence-response curve for this inhibition showed that blue light was maximally effective at fluence rates which exceeded 3 to 57 times that of the far-UV. Tonic red light, which was given from above, abolished to a large extent the antagonistic action of blue light. With a regimen of unilateral irradiation, i.e. when far-UV and blue light were given from the same side, a phototropic balance could be achieved with approximately equal fluence rates of blue and UV light. Above or below this critical balance point the bending was either negative or positive. In this setup the effect of tonic red light was complex. First, it caused an enhancement of the positive or negative bending, and second, it caused at some fluence rates a sign reversal from positive to negative phototropism. The balance point itself was only marginally affected. The data cannot be explained on the basis of a single photoreceptor and support the previous notion of separate far-UV and blue-light receptors. The antagonism between these two receptors probably occurs on the level of a red-light-absorbing receptor intermediate. Received: 16 November 1997 / Accepted: 18 December 1997     

From the salt marsh to the ID-card: Bacteriorhodopsin as a functional material in nanobiotechnology

Bacteriorhodopsin (BR) is an evolutionary highly optimized photochromic retinal protein, which is found in extremely halophilic bacteria, e.g., in salt marshes. We demonstrated that starting from the wildtype as a blueprint by means of gene technology and biotechnology a versatile material for optical information recording can be developed. BR is structurally related to the visual pigment rhodopsin. It is the key molecule in the halobacterial photosynthetic system — an alternative to the chlorophyll-dependent photosynthesis. Its biological function ist that of a light-driven proton pump. In the halobacterial cell — which are found e.g. in salt marshes — it converts light energy into chemical energy, i.e. a proton gradient over the cell membrane, which finally supplies ATP to the cell. The photochromic properties of BR are very attractive compared to those of known organic photochromic compounds, in particular as far as longevity under exposure to oxygen and light is concerned. This is one of the reasons why we try to utilized this evolutionary optimized biomaterial for technical applications in particular in optical data storage and processing. As the biological function of BR is optimized for energy conversion, the physical properties of BR need to be tuned to turn this molecule into a material which matches the requirements of optical applications in data storage and processing. Gene technology is a powerful tool for the controlled modification of physical properties of a biomolecule like BR. In technical applications water needs to be omitted. However, the function of biomaterials strictly depends on the presence of water. Membrane proteins are much less dependent on the presence of water which makes them good candidates for technical applications. We showed that BR can be processed into dry polymeric films where its function is preserved. In a field test where ID-cards comprising BR-based inks as security elements it has been demonstrated that biomaterials may be integrated in active form as functional components into conventional technical applications. Conventional nanomaterials supply properties to a product, biomaterials supply functions.